Objective To identify the rale of NKX2-5 gene in cardiomyocyte differentiation and its mechanism.Methods P19 cells were divided into transfected and non-transfected groups.In the transfected group,P19 cells were with stable expression of NKX2-5 gene.The P19 cells were cultured in suspension for 4 days,and the formed aggregates were transferred to Petri dish for adherent culture.On days 4,8,12,and 16 of the adherent culture,the expressions of ct-saicomeric actin(α-SA)and cardiac troponin T(cTnT)were detected with double-labeling immunofluorescence and Western blot.The ultrastruetural changes were observed on day 16.Results In the transfected group,no expression of α-SA and cTnT was found on day 4,and the expression of these 2 proteins or co-expression existed on days 8,12,and 16.There were early cell junction and myofilament-like structure in the cytoplasm of some cells in the transfected group.In the non-transfected group,these 2 proteins were negative,and no differentiated cell was found.Conclusion Stable expression of NKX2-5 gene can induce cardiomyocyte differentiation from P19 cells,but the P19 cells with stable expression of JVKX2-5 gene is not suitable to be an in vitro model of cardiac development.

Objective To analysis of the detection result of amniotic fluid chromosome which in NIPT high-risk pregnant women.Methods Amniotic fluid cells via amniotic cavity puncture were cultured and analyzed,the chromosome karyotypes were observed.Results The highest positive predictive value of NIPT was for trisomy 21(85.00%),then trisomy 18(75.00%),sex chromosome abnormalities(68.00%),other chromosome abnormalities(41.67%),trisomy 13 (25.00%).Conclusion The highest accuracy of NIPT was shown in detection of Down''s syndrome by NIPT.NIPT was screening test which is effective and noninvasive in prenatal diagnosis.Amniotic fluid Chromosomal karyotype analysis was the gold standard in the diagnosis of fetal chromosomal disease.

Objective: To investigate the change of autophagy level of bone marrow nucleated red blood cell (RBC) in patients with myelodysplastic syndromes (MDS) . Methods: Fifty-four MDS patients and thirty-three controls were enrolled in this study. The mitophagy were observed by transmission electron microscopy (TEM) . The level of autophagy-associated protein LC3B in GlycoA⁺ nucleated RBC was measured by flow cytometry. The expressions of ULK1 and mTOR mRNA in GlycoA⁺ nucleated RBC were measured by real-time PCR. The expression of the mitochondrial outer membrane protein TOM20 in GlycoA⁺ nucleated RBC was detected by Western blot. Results: Autophagosomes or autolysosomes were scarcely observed by TEM in MDS patients. The expression of LC3B in GlycoA⁺ nucleated RBC in high-risk MDS patients (0.22±0.12) was significantly lower than that in normal controls (0.43±0.22, P<0.001) , and lower than that in low-risk MDS patients (0.40±0.16, P=0.001) . The expression of AMPK [0.26 (0.60) ] in GlycoA⁺ nucleated RBC in high-risk MDS patients was significantly lower than that in controls [1.00 (2.07) , P<0.017) . The expression of ULK1 mRNA in GlycoA⁺ nucleated RBC in high-risk MDS patients [0.27 (3.31) ] was significantly lower than that in controls [1.07 (4.41) , P<0.017]. The level of mTOR mRNA in GlycoA⁺ nucleated RBC in high-risk MDS patients [1.82 (3.74) ] was significantly higher than that in controls [1.26 (1.38) , P<0.017]. The level of LC3B in GlycoA⁺ nucleated RBC was negatively correlated with the HGB (r=0.529, P=0.009) in high-risk MDS patients. The expression of mitochondrial outer membrane protein TOM20 in high-risk MDS patients was 9.42±4.42. Conclusion Autophagy is impaired in nucleated RBC of MDS patients.

Objective: To explore characteristic and function of peripheral blood mononuclear cells (PBMNC) -induced macrophages in patients with myelodysplastic syndrome (MDS) to couple with its progression. Methods: A total of 24 MDS patients (11 low-risk patients and 13 high-risk group patients) referred to Department of Hematology of Tianjin Medical University General Hospital and normal controls were enrolled from September 2014 to December 2015. PBMNC was stimulated with GM-CSF to transform to macrophages. The morphology of macrophages was observed by microscope. The quantity of macrophages, CD206 and SIRPα on surface of macrophages were detected by flow cytometry. The phagocytic function of macrophages was analyzed by fluorescence microscopy and flow cytometry. Results: The morphology of macrophages from MDS patients was abnormal. The percentage of transformed macrophages was (5.17±3.47) % in patients with MDS, which was lower than that in controls significantly[ (66.18±13.43) %, t=3.529, P=0.001]. The expression of CD206 on macrophages from MDS patients was significantly lower than that of controls[ (9.73±2.59) % vs (51.15±10.82) %, t=4.551, P<0.001]. The SIRPα level of macrophages from MDS patients was significantly lower than that of controls [ (0.51±0.09) % vs (0.77±0.06) %, t=2.102, P=0.043]. The phagocytic index and the percentage of phagocytic of macrophages from MDS patients were significantly lower than those of macrophages from normal controls[0.45±0.08 vs 0.92±0.07, t=-6.253, P=0.008; (23.69±3.22) % vs (42.75±2.13) %, t=-6.982, P=0.006 respectively]by flow cytometry. The phagocytic index of MDS patients was significantly lower than that of controls (0.24±0.04 vs 0.48±0.96, t=3.464, P=0.001) by fluorescence microscopy. Conclusion The quantity, recognization receptors and phagocytosis of PBMNC-induced macrophages decreased in MDS patients.

Programmed death-1 and programmed death-ligand 1(PD-1/PD-L1)are regulatory immune checkpoint molecules that inhibit T cell activation and,therefore,play an important role in tumor immunotherapy.In recent years,increasing numbers of targeted therapeutic agents have been developed,but single immune checkpoint blockers cannot completely inhibit tumor occurrence,and tumor escape sporadically occurs.Consequently,combination therapy of targeted drugs is considered a useful method to inhibit tumorigenesis and tumor development.T cell immunoglobulin and immunoreceptor tyrosine-based inhibition motif(ITIM)domain(TIGIT)is an inhibitory type 1 poliovirus receptor that has recently been a hotspot of targeted drug therapy research.It has been shown that the combination therapy of TIGIT plus PD-1/PD-L1 can reduce tumor escape and inhibit tumorigenesis more effectively.Therefore,this review summarizes and discusses the progress on the dual blockade of TIGIT and PD-1/PD-L1 pathways in tumor immunotherapy to provide a theoretical basis for tumor im-munotherapy.

Objective : To investigate the effects of luteolin on invasion and migration of endometriosis stromal cells and whether its mechanism is related to the regulation of 15-hydroxyprostaglandin dehydrogenase (HPGD) expres- sion． Methods : The endometrial stromal cells HEM15A were divided into control group (cells were cultured in nor- mal medium) ，luteolin group ( cells were treated with different concentrations of luteolin) ，si-HPGD group ( cells were transfected with si-HPGD) ，si-NC group ( cells were transfected with si-NC ) ，luteolin + si-HPGD Group (cells were transfected with si-HPGD and treated with 20 μmol / L luteolin) ，Luteolin + si-NC Group ( cells were transfected with si-NC and treated with 20 μmol / L luteolin) . Real-time quantitative PCR was used to detect mRNA level of HPGD．Cell proliferation was detected by CCK-8 assay，Transwell and scratch assays were used to detect cell invasion and migration．The protein expression of proliferating cell nuclear antigen (PCNA) ，matrix metallopro- teinase (MMP-2) ，MMP-9 and HPGD were detected by Western blot，and the level of prostaglandin E2 ( PGE2) was detected by ELISA. Results : Compared with 0 μmol / L luteolin，luteolin at 20，50 and 100 μmol / L significantly inhibited the proliferation activity of hEM15A cells，and reduced PCNA expression ( all P＜0. 05) ．Compared with the control group，20 μmol / L of luteolin significantly inhibited cell invasion and migration (P ＜0. 05) ，decreased the expressions of MMP-2 and MMP-9 (P＜0. 05) ，and up-regulated the mRNA and protein levels of HPGD (P < 0. 05) ，while inhibited cellular PGE2 level (P＜0. 05) ．Compared with the luteolin group，the luteolin + si-HPGD group increased cell invasion and migration (P ＜0. 05 ) ，increased the expressions of MMP-2 and MMP-9 (P < 0. 05) ． Conclusion Luteolin regulates HPGD / PGE2 signaling pathway to inhibit the invasion and migration of en- dometrial stromal cells.

A transient ischemic attack (TIA) can cause reversible and delayed impairment of cognition, but the specific mechanisms are still unclear. Annexin a1 (ANXA1) is a phospholipid-binding protein. Here, we confirmed that cognition and hippocampal synapses were impaired in TIA-treated mice, and this could be rescued by multiple mild stimulations (MMS). TIA promoted the interaction of ANXA1 and CX3CR1, increased the membrane distribution of CX3CR1 in microglia, and thus enhanced the CX3CR1 and CX3CL1 interaction. These phenomena induced by TIA could be reversed by MMS. Meanwhile, the CX3CR1 membrane distribution and CX3CR1-CX3CL1 interaction were upregulated in primary cultured microglia overexpressing ANXA1, and the spine density was significantly reduced in co-cultured microglia overexpressing ANXA1 and neurons. Moreover, ANXA1 overexpression in microglia abolished the protection of MMS after TIA. Collectively, our study provides a potential strategy for treating the delayed synaptic injury caused by TIA.
Animals ; Annexin A1/metabolism* ; CX3C Chemokine Receptor 1/metabolism* ; Chemokine CX3CL1 ; Cognition ; Dendritic Spines/metabolism* ; Ischemic Attack, Transient ; Mice ; Microglia/metabolism*

OBJECTIVE: To analyze the 28-day survival status and influencing factors of adult patients with sepsis, providing reference for early diagnosis of sepsis prognosis and reducing sepsis mortality. METHODS: A retrospective cohort study was conducted. A total of 160 adult patients with sepsis in the department of intensive care unit of the 940th Hospital of Joint Logistic Support Force of PLA from January 2021 to December 2022 were enrolled. The general information, laboratory examination results within 24 hours after admission, clinical treatment measures, and prognosis of patients were collected. Univariate analysis and binary multivariate Logistic regression were performed to screen the risk factors that might affect the short-term prognosis of patients with sepsis. Receiver operator characteristic curve (ROC curve) was plotted to analyze the predictive value of various risk factors on the short-term death risk of sepsis patients. RESULTS: A total of 160 patients with sepsis were enrolled, of whom 91 survived in 28 days, and 69 died with a mortality of 43.12%. Compared with the survival group, the patients in the death group were older, more severe, had higher blood lactic acid (Lac) level, and had more complications such as hypertension and multiple organ dysfunction syndrome (MODS). A total of 22 related factors were statistically significant: age, acute physiology and chronic health evaluation II (APACHE II) score, sequential organ failure assessment (SOFA) score, length of hospital stay, Lac, interleukin-6 (IL-6), fibrinogen (FIB), international normalized ratio (INR), ratio of prothrombin time (PT) to healthy people, prothrombin activity (PTA), PT, thrombin time (TT), oxygenation index (PaO₂/FiO₂), aspartate aminotransferase (AST), ratio of AST to alanine amninotransferase (ALT), serum creatinine (SCr), blood urea nitrogen (BUN), site of infection, history of hypertension, concurrent MODS, implementation of continuous renal replacement therapy (CRRT), and treatment with vasoactive drugs. Combined with the results of the univariate analysis, variables that might affect the short-term prognosis of septic patients were included in the multivariate Logistic regression analysis. The results showed that APACHE II score ≥ 20 [odds ratio (OR) = 1.106, 95% confidence interval (95%CI) was 1.003-1.219], Lac ≥ 5 mmol/L (OR = 1.430, 95%CI was 1.041-1.964), combined with hypertension (OR = 13.879, 95%CI was 1.082-178.016), concurrent MODS (OR = 98.139, 95%CI was 18.252-527.672) was an independent risk factor for 28-day death in adult septic patients (all P < 0.05). ROC curve analysis showed that the combination of the four indicators including APACHE II score, Lac, combined with hypertension, concurrent MODS, had predictive value for short-term outcomes in patients with sepsis. The area under the ROC curve (AUC) was higher than that of the 4 indicators alone [AUC (95%CI): 0.952 (0.918-0.986) vs. 0.745 (0.670-0.820), 0.816 (0.748-0.883), 0.608 (0.518-0.699), 0.868 (0.810-0.927)], the sensitivity was 94.2%, and the specificity was 90.1%. CONCLUSIONS APACHE II score within 24 hours, Lac, combined with hypertension, and concurrent MODS are independent risk factors for short-term mortality in adult septic patients in ICU. The combination of these indicators can make meaningful early clinical judgments on the short-term prognosis of septic patients, thereby reducing the mortality.
Adult ; Humans ; Retrospective Studies ; ROC Curve ; Sepsis/diagnosis* ; Prognosis ; Intensive Care Units ; Risk Factors ; Hypertension

OBJECTIVETo investigate the change of autophagy level of bone marrow mononuclear cells（BMMNCs）in patients with myelodysplastic syndromes（MDS）.

METHODSThirty- eight patients with MDS and 26 megaloblastic anemia patients were enrolled in this study. The autophagic vacuoles were observed by transmission electron microscopy （TEM） and the quantity of autophagic vacuoles was detected by monodansylcadaverine （MDC） staining. The LC3 protein positive cells were counted by immunofluorescence assays. The expression of Beclin 1, LC3A, mTOR mRNA were measured by real time PCR. The expression of Beclin 1 proteins were detected by Western blotting.

RESULTSThe autophgic vacuoles of double membrane that surrounds lysosomes appeared in MDS patients. The percentage of MDC positive cells was significantly higher in MDS patients［（9.75±2.63）%］than that of controls［（2.90± 0.89）%, P＜0.05）. The percentage of LC3 protein cells was also increased in MDS patients（6.13±1.03）% vs（1.5±0.58）%, P＜0.05）. The expression of Beclin 1 and LC3A mRNA in low-risk and intermediate-1 MDS were higher compared with controls （3.61 ± 3.02 vs 1.55 ± 1.03 and 6.56 ± 3.97 vs 1.21 ± 0.95 respectively, both P＜0.05）. The expression of mTOR mRNA was down- regulated in low- risk and intermediate-1 MDS compared with controls（0.39±0.37 vs 1.50±1.03, P＜0.05）. There were no significant difference in expression of Beclin 1, LC3 and mTOR mRNA among intermediate-2 and high-risk MDS and controls. Beclin 1 protein expression was higher in low- risk and intermediate- 1 MDS patients（1.257 ± 0.197）than that of controls（0.528±0.086）and inermediate-2 and high-risk MDS patients（0.622±0.118）.

CONCLUSIONThe autophagy levels were increased in low- risk and intermediate- 1 MDS, while not enhanced in intermediate-2 MDS. Autophagy might be considered as a cell protective mechanism in MDS. The relatively defective autophagy in intermediate- 2 and high- risk MDS might contribute to disease's progression.

Apoptosis Regulatory Proteins ; metabolism ; Autophagy ; Beclin-1 ; Bone Marrow Cells ; cytology ; Humans ; Membrane Proteins ; metabolism ; Microscopy, Electron, Transmission ; Microtubule-Associated Proteins ; metabolism ; Myelodysplastic Syndromes ; pathology ; TOR Serine-Threonine Kinases ; metabolism ; Vacuoles ; ultrastructure